Chaperoned ubiquitylation--crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex

CHIP is a dimeric U box E3 ubiquitin ligase that binds Hsp90 and/or Hsp70 via its TPR-domain, facilitating ubiquitylation of chaperone bound client proteins. We have determined the crystal structure of CHIP bound to an Hsp90 C-terminal decapeptide. The structure explains how CHIP associates with either chaperone type and reveals an unusual asymmetric homodimer in which the protomers adopt radically different conformations. Additionally, we identified CHIP as a functional partner of Ubc13-Uev1a in formation of Lys63-linked polyubiquitin chains, extending CHIP's roles into ubiquitin regulation as well as targeted destruction. The structure of Ubc13-Uev1a bound to the CHIP U box domain defines the basis for selective cooperation of CHIP with specific ubiquitin-conjugating enzymes. Remarkably, the asymmetric arrangement of the TPR domains in the CHIP dimer occludes one Ubc binding site, so that CHIP operates with half-of-sites activity, providing an elegant means for coupling a dimeric chaperone to a single ubiquitylation system.     

CHIP attenuates lipopolysaccharide-induced cardiac hypertrophy and apoptosis by promoting NFATc3 proteasomal degradation

Carboxyl-terminus of Hsc70 interacting protein (CHIP) is a chaperone-dependent E3-ubiquitin ligase with important function in protein quality control system. In the current research endeavor, we have investigated the putative role of CHIP in lipopolysaccharides (LPS)-induced cardiomyopathies. Basically, H9c2 cardiomyoblasts were transfected with CHIP for 24 hr, and thereafter, treated with LPS for 12 hr. Concomitantly, western blot analysis, actin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and coimmunoprecipitation studies were performed to investigate the underlying intricacies. Interestingly, western blot analysis revealed that the expression of hypertrophy and apoptosis-related proteins were considerably reduced following overexpression of CHIP. Moreover, Actin staining and TUNEL assay further ascertained the attenuation of cardiac hypertrophy and apoptosis following overexpression of CHIP respectively. These aspects instigate the role of CHIP in attenuation of LPS-induced cardiomyopathies. Additionally and importantly, co-immunoprecipitation and western blot studies revealed that CHIP plausibly promotes degradation of nuclear factor of activated T cells 3 (NFATc3) through ubiquitin-proteasomal pathway. Taken together, our study reveals that CHIP attenuates LPS-induced cardiac hypertrophy and apoptosis perhaps by promoting NFATc3 proteasomal degradation.     

序列特异的双链RNA可解除CHIP对TGF-β信号通路的抑制作用

根据热休克蛋白Hsc70C端结合蛋白CHIP(carboxylterminusofHsc70 interactingprotein ,CHIP)cDNA上特异序列合成寡核苷酸片段 ,以pBS U6为载体 ,构建产生CHIPdsRNA (doublestrandedRNA ,dsRNA)的表达质粒pBS U6 CHIPi .免疫印迹和免疫染色结果显示 ,pBS U6 CHIPi可以高效、特异地抑制哺乳动物细胞内CHIP基因的表达 .为进一步研究CHIP在TGF β信号通路中的调控作用 ,利用pBS U6 CHIPi从细胞水平上封闭CHIP基因表达 ,观察TGF β介导的基因转录活性的变化 .荧光素酶报告分析 (luciferaseassay)结果显示 ,CHIP明显抑制TGF β介导的下游基因的转录活性 ,而转染pBS U6 CHIPi能有效解除CHIP的抑制作用 ,从而提高TGF β信号通路转录水平 ,从逆向角度进一步验证了CHIP在TGF β信号途径中可能具有抑制性的调控功能     

Mutation of Gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3C-like protease: crystal structure with molecular dynamics simulations

SARS-CoV 3C-like protease (3CL(pro)) is an attractive target for anti-severe acute respiratory syndrome (SARS) drug discovery, and its dimerization has been extensively proved to be indispensable for enzymatic activity. However, the reason why the dissociated monomer is inactive still remains unclear due to the absence of the monomer structure. In this study, we showed that mutation of the dimer-interface residue Gly-11 to alanine entirely abolished the activity of SARS-CoV 3CL(pro). Subsequently, we determined the crystal structure of this mutant and discovered a complete crystallographic dimer dissociation of SARS-CoV 3CL(pro). The mutation might shorten the alpha-helix A' of domain I and cause a mis-oriented N-terminal finger that could not correctly squeeze into the pocket of another monomer during dimerization, thus destabilizing the dimer structure. Several structural features essential for catalysis and substrate recognition are severely impaired in the G11A monomer. Moreover, domain III rotates dramatically against the chymotrypsin fold compared with the dimer, from which we proposed a putative dimerization model for SARS-CoV 3CL(pro). As the first reported monomer structure for SARS-CoV 3CL(pro), the crystal structure of G11A mutant might provide insight into the dimerization mechanism of the protease and supply direct structural evidence for the incompetence of the dissociated monomer.     

An ADAR that edits transcripts encoding ion channel subunits functions as a dimer

In this report, we establish that Drosophila ADAR (adenosine deaminase acting on RNA) forms a dimer on double-stranded (ds) RNA, a process essential for editing activity. The minimum region required for dimerization is the N-terminus and dsRNA-binding domain 1 (dsRBD1). Single point mutations within dsRBD1 abolish RNA-binding activity and dimer formation. These mutations and glycerol gradient analysis indicate that binding to dsRNA is important for dimerization. However, dimerization can be uncoupled from dsRNA-binding activity, as a deletion of the N-terminus (amino acids 1-46) yields a monomeric ADAR that retains the ability to bind dsRNA but is inactive in an editing assay, demonstrating that ADAR is only active as a dimer. Different isoforms of ADAR with different editing activities can form heterodimers and this can have a significant effect on editing in vitro as well as in vivo. We propose a model for ADAR dimerization whereby ADAR monomers first contact dsRNA; however, it is only when the second monomer binds and a dimer is formed that deamination occurs.     

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.     

Key dimer interface residues impact the catalytic activity of 3CLpro,the main protease of SARS-CoV-2

3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited ∼60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro.     

Folded monomer of HIV-1 protease

The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR(R87K)) within the highly conserved Gly(86)-Arg(87)-Asn(88) sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR(R87K) produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg(87) destabilizes PR(R87K) by perturbing the inner C-terminal beta-sheet (residues 96-99 from each monomer), a region that is sandwiched between the two beta-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96-99) or both. Although both N- and C-terminal beta-strands were found to contribute to dimer stability, our results indicate that the inner C-terminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.     

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.     

Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR

The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor β receptor through transmembrane (TM) helix-helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively 15N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in 15N-(1)H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (ΔGx°=-3.05 kcal mol(-1)) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.     

Protein ubiquitination: CHIPping away the symmetry

CHIP is a ubiquitin ligase implicated in the degradation of misfolded proteins. In the November 23 issue of Molecular Cell, identified CHIP as a protein that interacts with the ubiquitin E2 complex Ubc13-Uev1A, which catalyzes the synthesis of Lys-63-linked polyubiquitin chains. Although the ubiquitin ligase activity of CHIP requires its dimerization through the U box domain, the crystal structure of the CHIP-E2 complex reveals that the protomers in the CHIP homodimer adopt distinct conformations such that only one U box of CHIP interacts with Ubc13.     

Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment

Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo.     

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders

The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross-functionality in additional neurodegenerative diseases, for instance, to eliminate polyglutamine-expanded proteins, reducing their aggregation and toxicity and reinstate proteasome function. The dual actions of CHIP (molecular co-chaperone and E3 ligase) and Parkin (as E3-ubiquitin ligase and anti-oxidant) may also play a role in suppressing inflammatory reactions in animal models of neurodegeneration. In this review, we focus on the significance of CHIP and Parkin as inducers of amyloid clearance, as cytoprotectants and in the suppression of reactive inflammation. A case is made for more effort to explore whether neurodegeneration associated with proteinopathies can be arrested at early stages by promoting their mutual action.